The goal of this project is to seek an understanding on a molecular basis as to the reason why certain metabolically important oxidation-reducton enzymes require an 8 alpha-substituted, covalently bound flavin coenzyme while others do not. The approaches to be used include a comparative study of redox and catalytic properties of the two cholesterol oxidases, one which has a normal FAD while the others contains an 8 alpha-N(1)- histidyl FAD. The dependence of oxidation-reduction potential on pH will be done on the cholesterol oxidases, thiamine oxidase and 8 alpha-substituted FMN-apoflavodoxin complexes and will be extended to mitochondrial succinate dehydorgenasewhich contains an 8 alpha-N(3)-histidyl FAD. Mechanistic studies utilizing steady-state and stopped-flow kinetic techniques will be done on liver monoamine oxidase; an enzyme of neuro-pharmacological importance in biogenic amine regulation which contains an 8 alpha-S-cysteinly FAD. Circular dichroism and ENDOR spectroscopic approaches will be used to monitor alterations in protein conformation on flavin reduction and probe the environments of the respective covalent flavin moieties in several enzyme systems. The role of the 8 alpha-substituent in one- electron transfer reactions will be probed by stopped-flow kinetic studies on reconstituted apoflavodoxin-8 alpha FMN complexes with cytchrome c. The results of these studies will be of importance in mitochondrial electron transport and in the elucidation of the mechanism of action of important enzyme systems such as succinate dehydrogenase and monoamine oxidase.